The overall goal of this project is to develop calibration techniques and reconstruction software (RS) capable of very high resolution for single photon emission computed tomography (SPECT) using pinhole collimation. Pinhole SPECT has the potential to be an excellent in vivo imaging modality for small-animal imaging because of its high sensitivity and large magnification for objects near the aperture. However, the reconstruction quality of pinhole SPECT degrades quickly when the system is not properly aligned mechanically and electronically. Our preliminary research has utilized radionuclide point sources to measure the alignment parameters. Laser alignment techniques will be developed and used to measure mechanical shifts with very high precision. Laser techniques will also be developed to measure the angular-dependent radius of rotation (ROR), which is one method to compensate for a moving axis of rotation. Tilt sensors will complement the radionuclide and laser information by allowing for the measurement of and compensation for angular-dependent tilts of the collimator and detector. Software will be developed and tested for including information from radionuclide sources, laser measurements, and tilt sensors into a global maximum likelihood fit. This software will include effects such as position-dependent pinhole sensitivity, projection width (including parallax), collimator tilt, and laser ROR correction. These effects will provide additional constraints on the fit. An integrated simulation will be developed to simultaneously predict radionuclide projections of sources, laser measurements, and tilt-sensor readings for user-selected calibration scenarios. This simulation will be an important tool for separating various real effects in an experimental scanner. It will generate simulated data to test the global fitting software. In addition, iterative RS will be extended to include the measured calibration information. We will develop and extend a technique for the RS to self-compensate for missing calibration information by maximizing the likelihood function with respect to that missing information using pinhole-specific models for acceleration. In addition, our radionuclide techniques will provide simultaneous information for further improvements to pinhole sensitivity and point-response models very near the aperture. These models are important for accurate quantification. Our goal is to develop the techniques and RS necessary for reconstruction resolutions in the range of approximately 0.5 mm or less.